Exposure to iron results in the manifestation of an inward short circuit current (Isc) in Caco-2 epithelia (Scott et al. 2002). This electrogenic pathway is consistent with DMT-1-mediated iron uptake. Tandy et al. (2000) have shown that DMT-1 expression changes with age in Caco-2 cells. This study investigated the relationship between developmental changes in DMT-1 mRNA expression and the magnitude of Fe2+-evoked Isc responses. Caco-2 cell monolayers were grown for 7, 14, 21 or 28 days before being voltage-clamped in Ussing chambers for Isc determinations. Cumulative concentration-responses were performed for iron in isotonic mannitol/HEPES buffer (37°C). Apical pH was 6.0, whilst basolateral pH was 7.4. Iron (iron-ascorbate; 1:10 molar ratio) was applied apically in concentrations ranging from 25-1000 μM. In parallel experiments, complementary DNA (cDNA) PCR primers for human DMT-1 IRE (iron regulatory element) and non-IRE were designed and first strand cDNAs were synthesised by priming with random hexamers. Reverse transcription was performed using DNase treated Caco-2 RNA at 7, 14, 21 and 28 days. Real-time PCR amplification and analysis was performed using a 7700 Sequence Detection System and SYBR Green Mastermix (Applied Biosystem). Standard curves were generated from cDNA (from total Caco-2 RNA). CT (cycle threshold) values were used to calculate linear regression lines to determine DMT-1 mRNA levels. After 7 and 14 days, Fe2+ failed to induce an Isc response. However, after 21 and 28 days, the Isc evoked by Fe2+ was concentration-dependent and saturable (day 21: KM = 238 ± 54 (4) μM and Vmax = 1.34 ± 0.28 (4) μA.cm-2; day 28: KM = 164 ± 20 (5) μM and Vmax = 1.00 ± 0.08 (5) μA.cm-2; mean ± SEM (n)). Caco-2 cells also demonstrated age-dependent expression of DMT-1 IRE mRNA (day 7 = 0.63 ± 0.19 (6); day 14 = 1.86 ± 0.23 (6); day 21 = 58.46 ± 20.5 (6); day 28 = 5.50 ± 1.17 (6), values expressed as DMT-1 mRNA (ng):18S mRNA (ng)). DMT-1 IRE mRNA expression was greater at day 21 than at days 7, 14 or 28 (P<0.001, one way ANOVA). There was no change in DMT-1 non-IRE mRNA levels. These data demonstrated that only DMT-1 IRE mRNA expression was time-dependent in Caco-2 cells. Maximal expression occurred around 21 days in culture and coincided with the maximum electrogenic response to iron in these cells. This is consistent with iron-evoked changes in Isc being due to apical Fe2+ uptake mediated by DMT-1.
|Title of host publication||Proceedings of The Physiological Society|
|Number of pages||1|
|Publication status||Published - 2005|
|Event||Physiological Society meeting 2005 - King's College London, London, United Kingdom|
Duration: 1 Jan 2005 → 1 Jan 2005
|Conference||Physiological Society meeting 2005|
|Period||1/01/05 → 1/01/05|